Vascular smooth muscle cell activation (VSMC) and phenotypical switch is critical to remodeling processes in vasculoproliferative disorders such as vein graft intimal hyperplasia, restenosis after endovascular interventions, cardiac transplant arteriopathy, pulmonary hypertension and additional obstructive diseases atherosclerosis and restenosis after percutaneus coronary intervention operations (1-5). The initiating events in the cellular activation are triggered by pertubations of the vessel wall and migratory and proliferative activity of VSMCs are key events in the pathologies and the interplay between the extracellular matrix (ECM) and integrins are essential in the control of hyperplasia. Outward remodeling processes may partly reduce the lumen loss, and are involving matrix breakdown by matrix metalloproteinases (MMPs) and result in increments of matrix breakdown products in the circulation.
MFAP4 is a 36 kDa glycoprotein composed of a short N-terminal region that contains a potential integrin binding RGD motiv followed by a fibrinogen related domain (FReD). The protein forms a homo-oligomeric structure under native conditions (6-8). FReDs are found in a diverse group of human proteins involved in different functions such as coagulation, angiogenesis, tissue growth and remodelling, and innate immunity (9).
Studies of MFAP4 or the bovine homologue MAGP-36 has been undertaken since 1989 (10). MAGP-36/MFAP4 was first identified as a protein with tenascin resemblance in the amino acid composition and localized to ECM in arteries (7, 11-14). MAGP-36 was following demonstrated with direct interaction with ECM fibers including elastin, collagen, or calvasculin (11-13, 15). The interaction between MAGP-36 and cellular integrin receptors was demonstrated using inhibition by RGD containing peptides of human aortic smooth muscle cells in attachment to immobilized MAGP-36 (12). All RGD dependent integrins may potentially interact with this RGD site, however integrins αVβ3/5 are highly relevant for investigation of vascular remodeling. Integrins αVβ3/5, are known to induce VSMC responses both in vivo and in vitro (16, 17) and may be upregulated during restenosis (18-25). The integrin αVβ3/5-dimer is expressed in the media in normal arteries (26), yet highly upregulated very early after injury (27, 28).
MFAP4 may be categorized as a matricellular protein due to the localization to matrix fibrils and interactions with smooth muscle cells. Other members of the matricellular family include e.g. tenascin-C; TSP-2 and -4, tenascin-X, and integrin αVβ3/5 ligands osteopontin, vitronectin, and periostin. Characteristically for such molecules, is that they are not essential for tissue homeostasis, whereas loss of the proteins is associated with a wide range of alterations in the remodeling tissues (29-31).
Besides the localization of MFAP4 in the vessels a soluble form of MFAP4 is present in broncho-alveolar-lavage and in serum (7, 32). Recent proteomic studies have shown MFAP4 to be upregulated in both liver fibrosis (32) and in lung tissue from patients with pulmonary arterial hypertension (33) suggesting that the expression or the turnover of the protein may reflect remodelling processes in diverse tissues.
Detailed mechanisms of progression of arteriosclerosis from pathogenesis to advanced disease are not sufficiently clarified. In addition, detailed mechanisms of vascular remodeling are also unknown. Although there are some reports describing relationships between angiotensin II receptor antagonists and vascular remodeling the effects of calcium channel blockers on pathological changes in arteriosclerosis and vascular damage as well as their mechanisms are little known. Furthermore, since percutaneous coronary intervention (PCI) including percutaneous transluminal coronary angioplasty (PTCA) and stent implantation have low invasiveness, they occupy the central position in current therapeutic strategies against ischemic heart diseases. However, restenosis appearing within several months after surgery in 30-45% patients undergoing these surgical procedures is a major problem. As for the mechanisms of restenosis following PCI, decreases in the diameters of whole vessels in the late period after PCI (that is, remodeling) are considered important, in addition to hyperplasia and hypertrophy of neointima caused by proliferation of smooth muscle cells and accumulation of extracellular matrix, which is produced by the smooth muscle cells. Under these circumstances, development of new medicaments that can effectively prevent restenosis of vessels following PCI is needed. Nevertheless, no medicaments with high efficacy have so far been developed.
Zhao et al (56) discloses human microfibril-associated protein 4 (MFAP4). Zhao et al further discloses that the N-terminus of the protein bears an Arg-Gly-Asp (RGD) sequence that serves as the ligand motif for the cell surface receptor integrin.
VASSILEV T. L. et al. (57) discloses antibodies against ligands to integrins, where the antibodies are against the RGD sequence, resulting in lack of ligand activation of the integrins
KOKUBO T. et al. (58) discloses that the blockade of the integrin αvβ3 by antagonists being either a blocking antibody to αvβ3 or a αvβ3-blocking RGD peptide reduced neointima by 70%. KOKUBO T. et al mentions vitronectin, fibronectin, osteopontin, fibrinogen and von Willebrand factor as ligands to αvβ3
Meanwhile, these prior art documents do not identify a specific receptor for the integrin MFAP4 or antibodies directed to MFAP4 thereby inhibiting the known functions of the integrin receptor. Based on the prior art it could not be predicted that MFAP4 had an effect.